ABSTRACT Sulfur mustard gas (SM) was first used as a chemical weapon in World War I and later in many conflicts including Syria in 2016. SM is a strong alkylating agent that rapidly penetrates into the cornea to disrupt the epithelial barrier, epithelial-stromal and basement membrane organization, and collagen fibril arrangement. This results in a grade 3 or 4 pathology clinically termed mustard gas keratopathy (MGK) that is characterized by severe ocular inflammation, recurrent corneal epithelial erosions and ulcerations, epithelial-stromal separation, and corneal fibrosis and neovascularization. MGK leads to progressive corneal degeneration, with biphasic acute and delayed-onset manifestations involving multiple mechanisms and signaling pathways. Our central hypothesis is that topical use of ophthalmic drops which inhibit inflammatory response, extracellular matrix (ECM) degradation and epigenetic modifications to the cornea can effectively treat acute MGK in vivo. We created a novel multimodal non-steroidal topical ophthalmic formulation, Turbo Eye Drop (TED), containing 4 FDA-approved drugs with differing mechanisms of action. Pilot studies demonstrated that TED is non-toxic to donor human corneal cells and potently mitigated toxicity of the nitrogen mustard (NM), a bi-functional SM analog, in human corneas ex vivo. The goal of the project is to test the novel hypotheses that (1) topical TED application effectively treats SM-induced corneal damage in vivo in rabbits without significant side effects, and (2) antidote properties of TED are due to significant inhibition of COX2 (cyclooxygenase-2), iNOS (inducible nitric oxide synthase), MMP9 (matrix metallopeptidase 9), TGF? (transforming growth factor beta) and VEGF (vascular endothelial growth factor) proteins. Two specific aims to test the hypotheses are: Aim-1 to examine the clinical efficacy and safety of topically applied TED (2 times daily for 5 days) to mitigate acute MGK in vivo using an established SM vapor injury rabbit model in collaboration with MRI Global in Kansas City, Missouri. This will be accomplished by performing in vivo clinical ophthalmic exams in rabbits following SM vapor exposure and studying corneal tissues collected after euthanasia by measuring inflammatory, angiogenic and fibrotic mRNA and protein levels, integrity of the basement membrane, epithelial-stromal organization and collagen fibril arrangement using qPCR, ELISAs, immunostaining, H&E, and transmission electron microscopy. We expect that TED will effectively treat MGK in vivo in rabbits without side effects. Aim-2 will elucidate underlying mechanisms and define utility of TED for humans using primary corneal cells, an ex vivo human organ culture model, H&E, qPCR, immunofluorescence, and ELISAs. We predict that TED will arrest NM-induced damage to human corneas by downregulating COX2, iNOS, MMP9, TGF?, and VEGF. Successful completion of this project will lead to the development of a novel first-line therapy for acute MGK and better understanding of the mechanisms driving SM toxicity in the cornea.